Insects cause significant pest problems in a wide variety of animals and plants worldwide, with estimates of 13% of crop production lost each year despite current control measures. Insect species of the orders Lepidoptera, Hemiptera, Orthoptera, Coleoptera, Psocoptera, Isoptera, Thysanoptera and Homoptera cause massive losses to many horticultural and broadacre crops and stored and manufactured grain products. Diptera, Anaplura, Malophaga and Siphonaptera cause parasitic infections in animals and man. Other orders (Hymenoptera, Dictyoptera, Isoptera) include important domestic and industrial pests.
Many of the known control measures for insects depend on the use of chemical insecticides, for example chlorinated hydrocarbons (DDT, endosulfan etc), organophosphates (chlorpyrifos, diazinon, malathion, parathion), organocarbamates (carbaryl, methomyl, proxypur) and synthetic pyrethroids (cypermethrin, deltamethrin).
Problems associated with the use of chemical insecticides include the development of resistance by target insects (organophosphates, synthetic pyrethroids), the persistence of the chemicals in the environment and in plant and animal tissues, and the harmful effects on non-target organisms (organochlorines, insect growth regulators).
Boron compounds (borax, polybor) have also been used for insecticidal purposes. Boron compounds are stable, kill insects relatively slowly at practical doses (Mullens and Rodriguez, 1992), and ingestion of large doses by humans can be lethal (Anon, 1991)
Other categories of insecticide include insect growth regulators (IGRs) and insecticidal bacterial toxins (eg. Bacillus thuringiensis (Bt) toxins). IGRs are compounds that interfere in some way with chitin synthesis. They include juvenile hormone analogues (methoprene), chitin synthesis inhibitors (fenoxycarb, diflubenzuron, flurazuron) and triazine derivatives (cyromazine). Resistance has been noted to many classes of IGR. Resistance is also developing in certain lepidoterans to Bt toxins. It is technically difficult with both IGRs and Bt toxins to ensure adequate insect kill at an appropriate stage in its life cycle. Some IGRs are stable and may pose environmental hazards.
The most useful groups of insecticides are those having high insecticidal activity and low environmental persistance (organophosphates, synthetic pyrethroids). The greatest problem associated with these, however, is the development of resistance by target insects. It is believed that 90% of insecticide use is still based on classical neurotoxic insecticides. The search for alternative low-residual insecticides which are effective on insects resistant to existing insecticides is thus particularly urgent.
Agents referred to as synergists may be used to maximise effectiveness of particular insecticides. Synergists may or may not be insecticidal in their own right. Blood and Studdert (1988) define a synergist as an agent that acts with or enhances the action of another. As an example, it may be noted that piperonyl butoxide is a synergist for synthetic pyrethroids. Synergists which are effective in combination with a particular insecticide may not be effective in combination with other insecticides. Synergists can be used to overcome problems of insect resistance, although insect resistance to synergists can also occur. The role of synergists in insecticidal formulations can be vital for achieving a commercially viable result, and for insecticide resistance management. The search for effective synergising combinations is as urgent as the search for effective insecticides per se (Forrester et al, 1993).
Recently attention has focused on insect peptidases, inhibition of which may provide a possible means of insect control. Peptidases are ubiquitous enzymes which break down proteins and peptides, and thus assist with digestion both in the gut and in cells. They are involved in tissue reorganisation during embryo development, moulting and pupation in insects. They are also involved with defence against invading organisms and with protein regulation.
Peptidases are a widely variable group of enzymes. Currently they are classified according to:
(1) the reaction catalysed PA1 (2) the chemical nature of the catalytic site, and PA1 (3) the evolutionary relationship as revealed by structure (Barrett, 1994) PA1 N-acetyl-L-phenylalanine ester (APNE) PA1 N-acetyl-L-tyrosine ethyl ester (ATEE) PA1 N-benzoyl-L-tyrosine ethyl ester (BTEE) PA1 N-benzoyl-L-tyrosine-p-nitroanilide (BTPNA) PA1 L-glytaryl-L-phenylalanine-p-nitroanilide (GPPNA) PA1 N-succinyl-L-phenylalanine-p-nitroanilide (SPAPNA) PA1 L-glutaryl-L-phenylalanine naphthylanide (GPNA). PA1 (a) effective inhibitors of the above category suitable for the genetic transformation of plants have not been identified, PA1 (b) the apparent role of these enzymes is minor relative to the dominant cysteine and/or serine peptidase activities in the gut of insects. PA1 (a) control of insect infestation by direct application to a plant or animal vulnerable to such infestation; PA1 (b) reduction of insect numbers by application of the agents of the invention to insect habitat or breeding sites; PA1 (c) control, either by way of prophylaxis or reduction in severity, of infections in plants or animals which are transmitted by insects; and PA1 (d) control, by way of prophylaxis or reduction in severity, of infections in plants or animals which are consequential upon insect infestation. PA1 1. Select representative active peptidases, which may be purified or in compositions comprising the active enzyme. PA1 2. Select an enzyme activity assay for each of the representative peptidases by selecting an appropriate substrate for the enzyme and conditions for the reaction so as to yield an appropriate quantifiable endpoint in a convenient time. PA1 3. The test compound is considered to be a peptidase inhibitor if a reaction inhibition of 50% or greater is found in any of the above enzyme activity assays. PA1 trypsin and chymotrypsin inhibitors, such as PA1 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride (PEFABLOC) PA1 serine or cysteine peptidase inhibitors derived from legumes, vegetables, fruits or cereals; PA1 cystatins and E-64; PA1 carboxypeptidase inhibitors from potato or other sources; PA1 Eglin C; PA1 L-leucinethiol. PA1 (a) The animals treated by the methods of the invention are selected from the group consisting of humans, sheep, cattle, horses, pigs, poultry, dogs and cats. PA1 (b) The plants treated by the methods of the invention are selected from the group consisting of cotton, oil seed crops, ornamental plants, flowers, fruit trees, cereal crops, vine crops, root crops, pasture plants and vegetables. PA1 (c) The insects to be controlled in the case of horticultural and broadacre applications of the invention are Lepidoptera, Hemiptera, Orthoptera, Coleoptera, Isoptera, Thysanoptera or Homoptera, in the case of insect infections in animals are Diptera, Anaplura, Malophaga or Siphonaptera, or in the case of domestic or industrial pests are Isoptera, Dictyoptera and Hymenoptera. PA1 (d) The transgenic plants are selected from the group consisting of cotton, oil seed crops, ornamental plants, flowers, fruit trees, cereal crops, vine crops, root crops, pasture plants, and vegetables, and PA1 (e) The transgenic organisms to be resident in or on an animal or plant are Bacillus sp or Pseudomonas sp or Mycobacteria sp. PA1 Preparing a suitable vector comprising a nucleotide sequence that codes for a peptide agent that inhibits an aminopeptidase or "non-strongly-chelating" peptidase and a promotor wherein the nucleotide sequence is capable of being expressed by a host contained the vector. PA1 Incorporating the vector into the host; and PA1 Maintaining the host containing the vector under conditions suitable for transcription and translation of the nucleotide sequence into said peptide agent.
The International Union of Biochemistry and Molecular Biology (IUBMC), In:Enzyme Nomenclature (1992), classifies peptidases by enzyme class (EC) categories. These categories are EC 3.4.11 to 3.4.19 for exopeptidases (those enzymes that only act near the ends of peptide chains) and EC 3.4.21 to 3.4.99 for enzymes that preferentially act on the inner regions of peptides. Group EC 3.4.99 is a group of peptidases for which the catalytic mechanism is unrecognised or uncharacterised.
An overview of the peptidase classes and their relationship to insect biochemistry is outlined below.
1. Serine Peptidases
This group includes serine-type carboxypeptidases EC 3.4.16, and serine endopeptidases EC 3.4.21.
Serine peptidases are typically recognised by a catalytically active serine amino acid at their active site, and by their sensitivity to an enzyme inhibitor, 3,4-dichloroisocoumarin (3,4-DCI). The preferred pH range for activity of mammalian serine peptidases is 2-8; however insect serine peptidases are commonly adapted to alkaline conditions (pH 9 to over 11 in some lepidopteran larvae).
The activity of serine peptidases in insects is also commonly defined by reaction of the enzymes with synthetic substrates. The three common categories of insect serine peptidases (trypsin-like, chymotrypsin-like and elastin-like) can be identified in this way. Trypsin-like serine peptidases react with synthetic substrates P-tosyl-L-arginine methyl ester (TAME), .alpha.-N-benzoyl-L-arginine ethyl ester (BAEE), .alpha.-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and benzoyl-DL-arginine naphthylamine (BANA).
Chymotrypsin-like peptidases may be identified by their reaction with:
Elastase-like serine peptidase inhibitors may be identified by their reaction with synthetic substrates such as N-succinyl-ala-ala-pro-leu p-nitroanilide (SAAPLpNA) (SEQ ID NO:1)
The activity of serine peptidases may also be described in terms of their reaction with enzyme inhibitors. Serine-group peptidases are generally inhibited by Di-isopropyl-fluro-phosphate (DipF/DFP), and paraphenyl methyl sulphonyl fluoride (PMSF). Trypsin-like peptidases are inhibited by tosyl-L-lysine chloromethyl ketone (TLCK). Chymotrypsin-like peptidases are inhibited by tosyl-L-phenylalanine chloromethyl ketone (TPCK). Elastase-like peptidases are inhibited by Eglin-C.
A number of naturally occurring proteins have been found to be able to inhibit serine peptidases. These include the crystalline soybean trypsin inhibitor of Kunitz (SBTI) and the soybean trypsin inhibitor of Bowman-Birk (BBTI). Various other legume seeds contain peptidase inhibitor at 1-4% of total protein, for example chickpea trypsin/chymotrypsin inhibitor (CI), Lima Bean trypsin inhibitor (LBTI) and cowpea trypsin inhibitor (CPTI) (MacIntosh et al, 1990). Animal-derived serine peptidase inhibitors include bovine pancreatic trypsin inhibitor (BPTI, Aprotinin), egg-derived ovomucoid and alpha-1-antitrypsin from blood.
Serine peptidases having alkaline pH optima are recognised to be of primary importance as soluble enzymes in the digestive fluids of insects. Examples include sphingidae (Miller et al, 1974), noctuidae (Ahmed et al. 1976, 1980; Ishaaya et al, 1971; Prichet et al, 1981; Teo et al, 1990; Broadway and Duffy, 1986), bombycidae (Sasaki and Suzuki, 1982; Euguchi and Iwanito, 1976; Euguchi and Kuriyama, 1985), pieridae (Lecadet and Dedonder, 1966; Broadway et al, 1989) pyralidae (Larocque and Houseman, 1990; Houseman et al, 1989; Mohammed and Altias, 1987) and diptera (Bowles et al, 1990). Cristeller et al (1992) showed that serine peptidases were implicated in the (casein) digestive activity of twelve phytophagous lepidopterans. In this article Christeller found that no other type of peptidase showed significant evidence of digestive activity. Serine peptidases have also been found to exhibit a dominant role in keratin-digesting lepidopterans (Christeller et al, 1994; Prowning and Irzykiewicz 1962; Ward, 1975 a, b). Furthermore, they have an important role in the digestive activity of some coleopterans (McGhie et al, 1995, Dymock et al, 1992), some orthopterans (Sakal et al, 1989; Christeller et al, 1990), some heteropterans (Cohen 1993) and in some dipterans (Bowles et al, 1990).
The digestive serine peptidases vary considerably both in number and in catalytic properties within and between species (Applebaum, 1985). In some instances inhibitors of serine peptidases in insect diets have been recognised to cause feeding deterrence as well as digestive inhibition (Dymock et al, 1992).
Trypsin-like serine peptidases have been recognised to be involved in the key growth regulatory area of molting. They exhibit several roles including process control, exposure of chitin fibrils to chitinase enzymes and in recycling of cuticular material (Samuels and Paterson, 1991).
Because of their dominant metabolic roles, their common natural occurrence, and the occurrence of many natural inhibitors in plants and animals, serine peptidases have received the most attention as agents for insect control. It is important to note that the context for use of serine peptidases in insect control has almost entirely been in the area of transgenic plants.
2. Cysteine (Thiol) Peptidases
This group includes cysteine-type carboxypeptidases (EC 3.4.18) and cysteine endopeptidases (EC 3.4.22).
These enzymes are characterised by possession of a catalytically active cysteine residue at their active site and by their sensitivity to certain inhibitors. Cysteine peptidases are characteristically activated under reducing conditions (added cysteine, dithiothreitol or other reducing agents). Cysteine peptidases are soluble enzymes generally found in midgut contents of insects. Mammalian cysteine peptidases commonly function in low pH conditions, although in insects, near neutral or mildly acidic pH values are favoured (Wolfson and Murdock, 1987).
The activity of cysteine peptidases is exemplified by their reaction with synthetic substrates n-.alpha.-benzoyl-L-arginine-p-nitroanilide (BAPNA) or benzoyloxy carbonyl-phe-arg-7-(4 methyl) coumaryl amide (Z-Phe-Arg-MCA). The activity of cysteine peptidases may also be described in terms of their reaction with enzyme inhibitors such as iodoacetamide, iodoacetate, heavy metals, p-chloromercuribenzoate, cystine cyanide, N-ethyl maleimide and characteristically, E-64. E-64 is a small peptide (L-trans-epoxy-succinyl-leucylamido-(4-guanidino-butane) obtained from a strain of Aspergillus japonicus. Naturally occurring cysteine peptidase inhibitors have been identified in microbes (E-64), plants (oryzacystatins I and II from rice grains and potato multicystatin (PMC) from potato tubers), and animals (hen-egg cystatin, HEC). Antipain is another well-known cysteine peptidase inhibitor.
Cysteine peptidases play a major role as soluble digestive enzymes of the gut of some insects, notably coleopterans (Orr et al, 1994; Thie and Houseman, 1990; Liang et al, 1991). The use of cysteine peptidase inhibitors for the control of insects has been largely explored in the context of transgenic plants (Orr et al, 1994; Wolfson and Murdock, 1987).
3. Aspartic Peptidases (EC 3.4.23)
These enzymes are typically recognized as possessing two aspartyl residues at the active site. Many aspartic peptidases are most active at low-pH values. Synthetic substrates for these enzymes include N-carbobenzoxy glutamyl-L-tyrosine and N-acetyl-L-phenanyl diiodotyrosin. Characteristic inhibitors for aspartic peptidases include pepstatin and diphenyl diazomethane (McDonald, 1985). Pepstatin is a naturally occurring inhibitor from a microbial source.
Applebaum (1985) has suggested some significance of aspartic peptidases to dipterans. Wolfson and Murdock (1987) have demonstrated some growth inhibition of a coleopteran (Colorado potato beetle larvae) by pepstatin; however, greater inhibition of growth was obtained by targeting cysteine peptidases.
An extensive search of the literature on peptidases has shown that only a limited amount of research has been conducted on insect aspartyl peptidases for insect control, either by transgenic modification of plants or by topical application. Christeller et al (1992) found that aspartic peptidase activity is apparently not evident in phytophagous lepidopteran gut material.
4. Metallopeptidases
These enzymes are typically recognised as possessing a catalytically active metal ion (commonly zinc) at the active site, and by their sensitivity to chelating agents. The metallopeptidase category includes some endopeptidases (enzymes that cleave within the peptide chain) and exopeptidases (enzymes that cleave amino acid(s) from the termini of peptides). Exopeptidases can further be categorised as carboxypeptidases (which cleave amino acid(s) from the C terminus) or aminopeptidases (which cleave amino acids from the N terminus).
Metallo endopeptidases (EC 3.4.22)
These enzymes have not been implicated in insect biochemistry, except for a possible role in wool digestion by keratinophagous lepidopterans (Prowning and Irykiewicz, 1962; Ward, 1975 a and b; Christeller et al, 1990, 1994).
Metallo carboxypeptidases (EC 3.4.17)
Metallo carboxypeptidases require a bivalent cation (usually Zn.sup.2+) for activity. They exist in both free and membrane-bound forms and favour activity at high (8-10) pH. Synthetic substrates for carboxypeptidases include hippuryl-DL-phenyl-lactic acid and hippuryl-L-arginine, for carboxypeptidase type A and B, respectively. Carboxypeptidase A-like enzymes appear to be more common in insects, and have been found in orthopterans, coleopterans, dipterans and lepidopterans.
Synthetic inhibitors for metallo carboxypeptidases include 1,10-phenanthroline, ethylene diamine tetraacetic acid (EDTA), .beta.-hydroxyquinoline and phosphoramidon. A naturally occurring inhibitor for metallo carboxypeptidase is potato-derived carboxypeptidase inhibitor (PCPI).
Carboxypeptidase enzymes in both free and membrane-bound forms have been recognised in mid-gut material from lepidopteran larvae (Ferreira et al, 1994; Christeller, 1990). Christeller et al (1992) described these enzymes as expected components of the protein digestion system. Prior to the priority date of this application these enzymes were not apparently recognised as targets for transgenic modification of plants, let alone as components of topical control agents.
Aminopeptidases (EC 3.4.11-3.4.13)
Aminopeptidases hydrolyse amino acids from the N-terminus of peptide chains, and are generally classified according to their dependence on metal ions (Zn.sup.2+ or Mg.sup.2+). The best studied aminopeptidases are found in the digestive tracts of mammals in both membrane bound and soluble forms. Aminopeptidases are commonly named with a suffix letter designating their pH optima, acidic (A) basic (B) or neutral (N), or by their membrane bound (M) state or by the number and type of amino acids cleaved from peptide substrates. These names are not exclusive; thus a leucine aminopeptidase M or N is an enzyme that preferentially but not exclusively removes leucine from a peptide, is membrane bound and whose activity is optimal at neutral pH. Aminopeptidases show preferential hydrolysis of leucyl, arginyl, methionyl, aspartyl, alanyl, glutamyl, prolyl, valyl and cysteinyl residues; however, substrate specificity is usually broad, and also depends on the other residues in the peptide chain (Taylor, 1993 a and b). Most aminopeptidases are metallopeptidases, although a few bacterial enzymes are known not to require metal ions for activity (Taylor, 1993 a and b). There are apparently few natural inhibitors of aminopeptidase.
Insect aminopeptidases usually have alkaline pH optima and are generally inhibited by bestatin (Taylor, 1993) and phenanthroline (Barrett, 1994).
Substrate specificities are broad, as stated above, but typical substrates include leu-pNA, arg-pNA, met-pNA and pro-pNA.
Metallo aminopeptidases are inhibited by chelating agents (such as EDTA), which can either remove the metal ion from the peptidase or form an inactive complex with the enzyme (Terra & Ferriera, 1994). The action of a particular chelator will vary with the type of aminopeptidase as well as with the chelator's own structure.
Aminopeptidases have been also recognised as part of the insect complement of digestive peptides, and are thought to be involved in the terminal stages of protein digestion (Terra & Ferriera, 1994). Christeller et al (1990, 1992) has asserted that exopeptidases (including aminopeptidases) will be of little use for insect control because of the minor role of these enzymes in the digestion of dietary protein relative to serine and cysteine peptidases.
Insect larval growth retardation following exposure to specific leucine aminopeptidase (LAP) inhibitors has not been reported. Christeller et al (1990) have shown with crude field cricket gut extracts that although exopeptidase levels, particularly LAP, are significant in this insect, exopeptidases contributed only 16-20% of protein digestion. In Christeller's study, it was further found that in the presence of two effective serine peptidase inhibitors, the contribution of exopeptidases to protein digestion was greatly reduced. This was attributed to lack of oligopeptide chain ends to act as substrates for exopeptidases. This further indicated a minor role for aminopeptidases in insect control.
The above findings teach strongly away from the use of exopeptidase inhibitors (principally aminopeptidase inhibitors) as insect control agents, and teach particularly strongly away from the combined use of serine peptidase inhibitors and aminopeptidase inhibitors as insect control agents.
Apart from a digestive role, aminopeptidase activity has been demonstrated in the moulting fluid of the lepidopteran Manduca sexta (Jungries, 1979). However, again the role of aminopeptidases appears to be secondary to the role of serine (trypsin-like) peptidases. No naturally occurring inhibitors of aminopeptidases or metallopeptidases important in insect development have been reported as effective in retardation of moulting. None have been used to provide insect-resistant transgenic plants.
Theoretically there appears to be a high potential for using peptidase inhibitors to control insects which are resistant to chemical insecticides. Firstly, the mechanism of action of peptidase inhibition is different from the mechanism of action of organophosphates and synthetic pyrethroids. Secondly, the amino acid sequence of the active sites of peptidase enzymes appears to be highly conserved (Taylor, 1993), thus indicating a low potential for mutation without consequent loss of activity. Finally, in the event that insect resistance to a particular peptidase inhibitor occurs, this resistance should not carry over to other peptidase mediated metabolic events.
However, there are significant problems associated with the use of peptidase inhibitors for the control of insects. High use rates may be required, leading to a product which is not cost-effective. Furthermore, whilst the peptidase inhibitor may limit larval growth, it is possible that a plateau is reached in the dose-response curve, even at elevated use rates. Peptide-based or protein-based peptidase inhibitors, while presumably non-residual in the environment, may be difficult to store over extended periods, and may lose efficacy when delivered in hard water. Subtle issues involving the source of a particular enzyme inhibitor may need to be addressed. For example, anomalous responses involving growth inhibition of Costelytra zealandica by soybean, potato and cowpea trypsin inhibitor and growth stimulation by lima bean trypsin inhibitor have been noted (Dymock et al, 1992).
Considerable research has centred on biochemical investigations of insect protein metabolism, principally digestive metabolism, using insect tissues, gut extracts, semi-purified or purified enzymes in vitro.
Much of this work has been directed toward the selection of enzyme inhibitors for the transgenic modification of plants by investigating the relationship of digestive enzymes with protein or peptide based inhibitors (Applebaum, 1985; Christeller et al, 1989, 1990, 1992; Lenz et al, 1991; Teo et al, 1990; Pritchett et al, 1981, Santos and Terra, 1984; Dow, 1986; Sakal et al, 1984, 1989).
Relatively fewer studies demonstrate a direct adverse influence of peptidase inhibitors on insect growth and/or reproduction. Serine and cysteine peptidase inhibitors have been shown to reduce the larval growth and/or survival of various insects, including Callosobruchus macalatus, Leptinotarsa decemlineata, Heliothis spp, Spodoptera exiqua, Costelytra zealandica, Teleogryllus commodus, Diabrotica sp, Manduca sexta, red four beetle and bean weevil (Gatehouse and Boulter, 1983; Shuckle and Murdock, 1983; Shade et al, 1986; Wolfson and Murdock, 1987; Broadway and Duffey, 1986; Hilder et al, 1987; Dymock et al, 1992; Orr et al, 1994; Burgess et al, 1994; Hines et al, 1990).
Generally insect growth inhibition has been achieved with inhibitors of principal digestive enzymes of the gut, and a method for selection of appropriate insecticidal inhibitors based on these enzymes has been described by Christeller et al (1992).
None of the above studies appears to be directly aimed at the production of topical insect control agents through interference with protein metabolism. In International Patent Application No. W094/16565 by Czapla, however, a minor claim cites the topical use of aprotinin or another serine peptidase inhibitor with 90% homology to aprotinin for control of the European corn borer (ECB) and Southern corn rootworm (SCR). It was claimed that aprotinin could be used alone or in combination with an insecticidal lectin. Czapla found that incorporation of aprotinin at 20 mg/ml of diet killed 100% of neonatal ECB larvae in a laboratory assay, and killed 60% of neonatal SCR. Ingestion rates as high as these would be difficult to achieve by topical application, and treatment costs would be unlikely to be competitive with chemical insecticides. Czapla found that the serine peptidase inhibitor SBTI (Kunitz and Bowman-Birk) and the cysteine peptidase inhibitor cystatin were less effective than aprotinin.
Direct feeding of SBTI to blood sucking insects has been investigated by Deloach and Spates (1980). They found raised mortality and suppressed egg hatch when SBTI was encapsulated in bovine erythrocytes and used as a bait for horn-fly. Various natural peptidase inhibitors (principally of serine peptidases) are known in blood. However, they have limited efficacy in protecting the animal from insect attack (Sandeman et al, 1990).
Wolfson and Murdock (1987) observed that whilst there is extensive documentation on the presence and distribution of peptidase inhibitors in plants, and these inhibitors are presumed to be targeted at insect digestive peptidases, there is little direct evidence to support their efficient action in inhibiting insect growth and development. These authors demonstrated that larval growth reduction in Colorado potato beetle could be obtained by feeding E-64 (a cysteine peptidase inhibitor) at threshold levels of 50 .mu.g/ml on potato leaves. However at a much higher application level (1000 .mu.g/ml) a plateau in mortality of 74-85% was found, which is insufficient for practical use. SBTIs (Kunitz and Bowman-Birk) were ineffective as growth retardants, and there was only a small response to pepstatin.
A research paper by Dymock et al (1992) has discussed the inhibition of growth of a larval coleopteran (New Zealand native grass grub) by peptidase inhibitors. The research was focussed on the genetic transformation of important pasture species, as the grubs feed on roots. Bioassays showed growth inhibition using serine peptidase inhibitors. Some anomalous responses to particular inhibitors were noted (inhibition by SBTI, POT I, POT II, CpTI, stimulation by LBTI). Cristeller et al (1989) had previously identified trypsin as the principal gut peptidase in the above grub, despite the fact that normally coleopteran gut peptidases are predominantly of the cysteine category. Generally the use rate of peptidase inhibitors required to achieve mortality was too high to be cost effective in topical use.
Compositions that function by inhibition of metallopeptidases (including aminopeptidase or LAP) have not been commercially developed for the control of insects. In fact, the prior art teaches away from the use of peptide-based aminopeptidase inhibitors or metallopeptidase inhibitors for insect control, because:
Shenvi (1993) has discussed the use of .alpha.-amino boronic acid derivatives as effective inhibitors of mammalian aminopeptidases. Shenvi notes that certain intermediates in the synthesis of .alpha.-amino boronic acid derivatives have insecticidal properties; however these intermediates did not have an amino group, and are not suggested to act either as aminopeptidase inhibitors or peptidase inhibitors of any sort.
The hexadentate metal chelating agent EDTA has been recognised by Samuels and Paterson (1995) and Ferreira and Terra (1986) to be an inhibitor of an aminopeptidase derived from the moulting fluid and digestive membranes. There is no recognition of any insecticidal effect of EDTA; however general claims for the insecticidal action of metal chelating agents have been made by Tomalia and Wilson (1985, 1986). No supporting evidence was presented. The use of metal chelating agents for insect control would be expected to be adversely influenced by the use of hard water for spray application, or if there was mineral or soil contamination of the materials to be treated.
We have now surprisingly found that compositions comprising an aminopeptidase inhibitor or metallopeptidase inhibitor and further comprising a non-strongly-chelating peptidase inhibitor are able to prevent the hatching of insect eggs and/or the development of insect larvae. The person skilled in the art will recognise that the vast majority of aminopeptidase inhibitors are in fact non-strongly-chelating, as this term is defined herein.
It will be clearly understood that the invention is applicable to the control of insects via a variety of mechanisms. The methods of the invention may, for example, result in the actual killing of insects, or in the interruption of insect growth and development so that maturation is slowed or prevented. Prevention of hatching of insect eggs is particularly desirable, since many economically important insects cause damage as a result of the feeding activities of their larvae.
It will be also understood that because of the wide variation of individual biochemical capacities within members of the class Insecta, responses to particular inhibitors and/or combinations of inhibitors will vary between species. Thus it is possible that some compositions within the scope of this invention will be poorly effective or even ineffective against some insects, while being highly effective against others. Variations in responses may also be seen at subspecies level or at different stages in the life cycle for particular insects, or even with the diet of the insects. Those skilled in the art will be able to match relevant inhibitors to insect targets by application of normal trial and error laboratory and field experimentation.
It will also be apparent to the person skilled in the art that the invention may be utilised in variety of ways, including but not limited to:
For the purpose of this specification, the term "peptidase inhibitor" is to be understood to be any compound able to inhibit any peptidase.
In practical terms a peptidase inhibitor can be identified by the following process.
Specifically, a metallopeptidase inhibitor or an aminopeptidase inhibitor may be identified in this way.
The term "non-strongly-chelating peptidase inhibitor" is to be understood to mean a peptidase inhibitor that chelates Zn.sup.2+ ions less strongly than EDTA in a competitive binding assessment. Both EDTA and the inhibitor should be at the same concentration in the reaction mixture, which may conveniently be 0.1 mM. The person skilled in the art will be able to choose appropriate reaction conditions and methods for determining Zn.sup.2+ distribution (eg. using multinuclear nuclear magnetic resonance (NMR), UV spectroscopy, voltametric techniques and soft ionization mass spectroscopy).